WO2022234536A1

WO2022234536A1 - Hydrogel based on hyaluronic acid polymer for use for treating solid tumours

Info

Publication number: WO2022234536A1
Application number: PCT/IB2022/054229
Authority: WO
Inventors: Helena Susana DA COSTA MACHADO FERREIRA; Rui Luís GONÇALVES DOS REIS; Nuno João MELEIRO ALVES DAS NEVES
Original assignee: Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies (A4Tec) - Associação
Priority date: 2021-05-06
Filing date: 2022-05-06
Publication date: 2022-11-10

Abstract

The present disclosure relates to a hydrogel composition for use in the treatment or therapy of a solid tumor comprising a hyaluronic acid polymer; one or more peptide sequence, wherein the peptide sequence is an inhibitor of extracellular matrix components, namely fibronectin, tenascin C, vitronectin, laminin or collagen; and liposomes, wherein the liposomes are arranged to physically crosslink the hyaluronic acid polymer; or hyaluronic acid and at least one peptide sequence. The disclosure also relates to a pharmaceutical composition for use in the treatment or therapy of a solid tumor, particularly gliomas and preferably glioblastoma, comprising a therapeutically effective amount of the disclosed hydrogel and a pharmaceutically acceptable excipient/carrier.

Description

D E S C R I P T I O N

HYDROGEL COMPOSITION FOR SOLID TUMORS

TECHNICAL FIELD

[0001] The present disclosure relates to a hydrogel composition for use in the treatment or therapy of solid tumors, in particular gliomas, such as glioblastoma, comprising hyaluronic acid, a peptide sequence inhibiting the bioactive signaling of extracellular matrix components, namely fibronectin, tenascin C, vitronectin, laminin or collagen, and liposomes physically crosslinking hyaluronic acid, wherein the liposomes also encapsulate an anti-cancer drug.

BACKGROUND

[0002] Solid tumors, according to the National Cancer Institute, are defined as "an abnormal mass of tissue that usually does not contain cysts or liquid areas." The most common solid tumors include brain tumors, sarcomas and lymphomas. Indeed, the central nervous system (CNS) can present several tumor subtypes, which are mainly diagnosed in the brain, but they can also occur in meninges, spinal cord, and cranial nerves [1] They can be primary (with origin in CNS cells) or secondary (cancer cells with a different origin that migrate to the CNS) tumors. Moreover, the World Health Organization (WHO) grades CNS tumors into a malignant scale from I to IV [2]. Particularly, gliomas that have their origin in glial cells or stem/progenitor cells represent =80% of all malignant brain tumors, in adults [3]. Moreover, a glioma classified at the first diagnosis at a low grade can evolve for more malignant types. For instance, diffusely infiltrating gliomas (grade II) can evolve to anaplastic astrocytomas or oligodendrogliomas (grade III) or to glioblastoma (GB), grade IV [4] Indeed, GB is the most frequent, malignant and lethal primary brain tumor. Patients have a very poor prognosis, with a median survival of 12-15 months after diagnosis [5], being the long-term survival practically inexistent (five- and ten-year survival rates of 5.5% and 2.9%, respectively [6]). This scenario clearly illustrates the absence of an effective treatment in the clinic for GB. The current standard therapy for GB was improved in 2005 [7] and remains unaltered until nowadays [8] It consists in maximal surgical resection of the accessible tumor, followed by radiotherapy and oral chemotherapy with temozolomide (TMZ). However, the efficacy of TMZ is highly dependent on the O- 6-methylguanine-DNA-methyltransferase (MGMT) expression in patients, resulting in the inhibition of the methylation of the MGMT promoter in patients with TMZ resistance [9] Moreover, as the complete surgical resection is unachievable by the difficulty in visualizing the tumor limits and the need to avoid excessive tissue removal, it is very difficult to avoid recurrence of the tumor. The resident cancer cells will also take advantage of the recovery period of the patient after surgery before initiating chemoradiotherapy [10] In addition to the rapid tumor growth, the proliferation of cancer stem cells and drug resistance mechanisms will also contribute for poor treatment outcomes. Indeed, the GB recurrences decrease the survival of the patients to less than 6 months [11]

[0003] The local delivery of anti-cancer drugs has shown potential to increase the survival rate of GB patients [12-16] For instance, OncoGel, a thermosensitive, biodegradable triblock copolymer composed of poly(lactide-co-glycolide) and polyethylene glycol) loaded with paclitaxel, demonstrated its safety after intracranial injection in rats. Moreover, it improved the survival in a rodent glioma model, mainly if combined with radiotherapy [17] TMZ loaded in a polyethylene glycol dimethacrylate (PEG-DMA) injectable and photopolymerizable hydrogel was able reduce the tumor weight on a xenograft U87MG tumor-bearing nude mice by inducing apoptosis only at the tumor center since proliferative cells were detected at its periphery [18] In another work, TMZ and paclitaxel incorporated in a polyethylene glycol- dipalmitoylphosphatidyle-thanoiamine (mPEG-DPPE) calcium phosphate nanoparticles (NPs) injectable thermoresponsive hydrogel (nanocomposite gel) showed promise for replacing free drug combinations after in situ administration in C6 tumor-bearing rats, but its safety was not evaluated [19] Paclitaxel in PLGA-NPs and TMZ coloaded in PEG- DMA hydrogels were safe, effective and synergistic in the local treatment of the U87MG orthotopic model [20] Indeed, this combination of drugs suppressed tumor growth more efficiently than the single drugs. Finally, a hydrogel of lauroyl- gemcitabine lipid nanocapsule was able to delay the formation of recurrences, but not avoided the death of 9L tumor-bearing resected rats, after brain administration [21]. Despite the promising therapeutic roles of local drug delivery, only Gliadel^® wafer (a carmustine implant) was approved by FDA for brain implantation [12, 22] Indeed, the bulk of FDA and EMA drug approvals (TMZ and recently bevacizumab, an anti- angiogenic antibody to target the high vascularization of GB) [23-26], reflects the difficulty of an effective and safe GB treatment. Moreover, Gliadel^® wafer has been combined with radiotherapy or chemotherapy to increase the overall survival of the patients [12, 27] However, that increase was not so pronounced as desired.

[0004] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

GENERAL DESCRIPTION

[0005] The present disclosure relates to a hydrogel composition for use in the treatment of a solid tumor, particularly gliomas, such as glioblastoma, comprising hyaluronic acid, a peptide sequence inhibiting the bioactivity of extracellular matrix components, namely fibronectin, tenascin C, vitronectin, laminin or collagen over the tumor cells, and liposomes physically crosslinking hyaluronic acid, wherein the liposomes encapsulate an anti-cancer drug.

[0006] In an embodiment, the injection or local administration, in the tumor resection cavity, of a formulation that allows the local and sustained release of anti-cancer drugs allows to start the treatment immediately after surgery. Moreover, the local administration has several advantages, such as maximizing local therapeutic efficacy and avoiding the limitations of a systemic drug administration, namely transport through the blood-brain barrier (BBB) and serious systemic side effects.

[0007] Hyaluronic acid (HA) is a polymer naturally present in the central nervous system (CNS) and one of the main structural components of the brain extracellular matrix. In an embodiment, HA hydrogels can assist brain repair and promote neural differentiation [28-30]

[0008] In an embodiment, HA was functionalized with a peptide, to increase the migration and adhesion of GB cells to the hydrogel, avoiding their invasion in the brain. The polymer chains of the functionalized HA are physically crosslinked by liposomes incorporating anti-cancer drugs. Indeed, liposomes are mixed with a noncovalent interaction to HA matrix, and consequently a physical reversible crosslinking of the hydrogel is obtained [31].

[0009] In an embodiment, the peptide is a fibronectin inhibitor peptide, such as SEQ ID No 1 (H-Arg-Gly-Asp-Ser-OH, RGDS) or SEQ ID No 2 (Gly-Arg-Gly-Asp-Ser-Pro-Lys), that interferes with the integrin-binding receptor of fibronectin. Fibronectin is an extracellular matrix (ECM) protein that is overexpressed in several cancers and influences tumor growth, invasion, metastasis and resistance to therapy [32] The inhibition of the fibronectin binding of cancer cells present benefits in cancer treatment, as the interaction of fibronectin inhibitor peptide, such as SEQ ID No 1 (a RGDS peptide), with cell surface integrins alters intracellular signaling pathways that can be beneficial for the treatment outcomes. In another embodiment, other peptides can be used to inhibit cancer cells integrins to ECM molecules e.g. vitronectin binding to cancer cells [34] In an embodiment, the peptides facilitate the attachment of the cancer cells to the hydrogel, avoiding their migration for other tissues of the brain, causing a great impact in therapy outcomes.

[0010] In an embodiment, fibronectin inhibitor peptide, vitronectin or another ECM inhibitor peptide can be selected from a list comprising the sequences listed in Table 1.

Table 1: Amino acid sequence listing for fibronectin, vitronectin, laminin, tenascin C and collagen inhibitor peptide.

[0011] In an embodiment, liposomes can be loaded with anti-cancer drugs selected from a list comprising drugs used in therapeutic, repurposing or promising drugs (e.g. doxorubicin -DOX, CXCR3-A inhibitors), immune checkpoint inhibitors (e.g. T- lymphocyte-associated antigen 4 - CTLA-4 - and programmed death 1 - PD-1 -, programmed death-ligand 1 - PD-L1 -, indolamine-2, 3-dioxygenase - IDO - and arginase 1) and natural compounds (e.g. dihydroartemisinin), alone or in combination. The repurposing drugs for GB can be selected from a list comprising mefloquine, ritonavir, ribavirin, chlorpromazine and ivermectin.

[0012] In an embodiment, the biocompatible and biodegradable hydrogel composition was designed for the direct injection or administration into the resection cavity, immediately after removal of the tumor in the surgery or injected intratumorally in non-operable GB tumors. In another embodiment, the composition presents a viscoelastic behavior that allows the desirable interaction with the surrounding tissues, avoiding empty spaces. Consequently, the hydrogel composition allows a sustained and gradual release of the incorporated drug(s) where it is needed. In an embodiment, the incorporated drug(s) was selected according to its undoubtedly efficacy as chemotherapeutics, to efficiently promote GB cells death. In another embodiment, being locally administered in a formulation, problems related with solubility, systemic side effects and poor brain penetration were overcome. Moreover, this strategy allows decreasing the dosage and, consequently, side effects. As this strategy avoids the time lapse between surgical resection and chemoradiotherapy onset, the proliferation of the remaining cancer cells is prevented.

[0013] The present disclosure relates to a hydrogel composition for use in the treatment or therapy of a solid tumor comprising a hyaluronic acid polymer; one or more peptide sequence, wherein the peptide sequence is an inhibitor of an extracellular matrix protein, such as fibronectin, tenascin C, vitronectin or laminin; and liposomes, wherein the liposomes are arranged to physically crosslink the hyaluronic acid polymer; or hyaluronic acid and at least a sequence 90% identical to the sequences of the following list: SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, or mixtures thereof, wherein the peptide sequence is chemically bound to the hyaluronic acid polymer. Preferably 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical or identical.

[0014] In an embodiment, the liposomes are used as drug delivery devices. In a further embodiment, the liposomes encapsulate an anti-cancer drug.

[0015] In an embodiment, the extracellular matrix protein is selected from a list comprising: fibronectin, tenascin C, vitronectin, laminin, collagen or mixtures thereof.

[0016] In an embodiment, the one or more peptide sequence comprises at least a sequence 90% identical to the sequences of the following list: SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, or mixtures thereof.

[0017] In an embodiment, the one or more peptide sequence comprises at least a sequence 95% identical to the sequences of the following list: SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, or mixtures thereof. Preferably 96% identical, 97% identical, 98% identical, 99% identical or identical.

[0018] In an embodiment, the hydrogel is for use in the treatment or therapy of glioblastoma. The uniqueness of this approach over current treatments is that it provides a matrix favourable for cancer cells attachment without the undesirable extracellular matrix signals for tumour growth and invasiveness, while allows a sustained and gradual release of drug(s) to efficiently kill them.

[0019] In an embodiment, the hydrogel composition of the present disclosure comprises: 1-20% (wt/Vh_ydro_gei) of hyaluronic acid, preferably 2-10% (wt/Vh_ydro_gei) of hyaluronic acid; 100 to 10000 ng/mL of the one or more peptide sequence, preferably 400 to 1000 ng/mL of the one or more peptide sequence; and 5-10000 mM of liposomes, preferably 50-1000 pM of liposomes; wherein the liposomes encapsulate 0.1-10000 pM of an anti-cancer drug, preferably 1-1000 pM of anti-cancer drug.

[0020] In an embodiment, the one or more peptide sequence is chemically bound to the hyaluronic acid polymer. [0021] In an embodiment, the bound between the hyaluronic acid and the peptide sequence is cleavable by proteolytic enzymes, such as matrix metalloproteinases.

[0022] In an embodiment, the liposomes are physically linked to hyaluronic acid by electrostatic interactions.

[0023] In an embodiment, the liposomes are small unilamellar liposomes, large unilamellar liposomes, or mixtures thereof.

[0024] In an embodiment, the liposomes have a size ranging from 25 to 400 nm, preferably from 110 to 130 nm.

[0025] In an embodiment, the liposomes are large unilamellar liposomes of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine, phosphatidylcholine (PC), phosphatidylserine, glycerophosphocholine, glycolipids, hydrogenated PC, cholesterol, PEGylated phospholipids (e.g. DSPE-PEG), PEGylated phospholipids containing a functional group at the polymeric end (e.g. DSPE-PEG-Mal; DSPE-PEG-NEh; DSPE-PEG- COOH; DSPE- l,2-Distearoyl-sn-glycero-3-phosphorylethanolamine; Mai - Maleimide), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and/or cholesteryl hemisuccinate (CHEMS).

[0026] In an embodiment the amount of anti-cancer drug in the hydrogel ranges from 0.1 to 10000 mM of the anti-cancer drug.

[0027] In an embodiment, the anti-cancer drug is selected from a list comprising drugs used in therapeutic, immune checkpoint inhibitors, natural compounds, repurposing or promising drugs, or mixtures thereof.

[0028] In an embodiment, the anti-cancer drug is selected from a list comprising T- lymphocyte-associated antigen 4, programmed death 1, programmed death-ligand 1, indolamine-2, 3-dioxygenase, arginase 1, dihydroartemisinin, doxorubicin, CXCR3-A inhibitors, mefloquine, ritonavir, paclitaxel, lomustine, carmustine, bevacizumab, ribavirin, chlorpromazine, temozolomide, ivermectin, or mixtures thereof. In a further embodiment, the anti-cancer drug is doxorubicin.

[0029] In an embodiment, the hydrogel is injectable or easily administrable in-situ or intra-tumor with a neurosurgical instrument, such as a spatula. [0030] In an embodiment, the hydrogel is administrable by an in-situ injection or intra tumor injection.

[0031] In an embodiment, the hydrogel composition allows a gradient of concentrations that facilitates the death of tumor cells in contact with the hydrogel, without compromising normal cells.

[0032] In an embodiment, the storage modulus at 37 °C of the disclosed hydrogel ranges from 0.5 to 3.5 kPa. In another embodiment, the storage modulus at 25 °C of the disclosed hydrogel ranges from 0.4 to 5 kPa.

[0033] An aspect of the present disclosure relates to a pharmaceutical composition for use in the treatment or therapy of a solid tumor, preferably glioblastoma, comprising a therapeutically effective amount of the disclosed hydrogel and a pharmaceutically acceptable excipient/carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0035] Figure 1: Embodiment of the size (A), PDI (B), and zeta potential (C) of LUVs for 10 days.

[0036] Figure 2: Embodiment of STEM images of empty LUVs (A) or incorporating DOX (B). Scale bar of 100 nm.

[0037] Figure 3: Embodiment of a DSC thermograms of LUVs incorporating or not DOX.

[0038] Figure 4: Embodiment of an ¹H-NMR spectra of RGDS, HA-RGDS and HA.

[0039] Figure 5: Embodiment of an FTIR spectra of hyaluronic acid (HA), RGDS and their chemical bonding (RGDS-HA).

[0040] Figure 6: Embodiment of elastic (G') and viscous (G") moduli of 1%, 2.5% and 5% (w/v) hyaluronic acid (HA) at 25 °C (A) and 37 °C (B) with 1% strain. [0041] Figure 7: Embodiment of elastic (G') and viscous (G") moduli of hydrogels of hyaluronic acid not (HA) or functionalized with RGDS (HA-RGDS) and without (HA+LUVs) or with (HA-RGDS+LUVs) LUVs at 25 °C (A) and 37 °C (B) with 1% strain.

[0042] Figure 8: Embodiment of DSC thermograms of hyaluronic acid (HA; A) and after functionalization with RGDS (B) and incorporating or not LUVs containing or not DOX.

[0043] Figure 9: Embodiment of SEM images of hydrogels of hyaluronic acid without (A) or with LUVs (B) and after its RGDS-functionalization without (C) or with LUVs (D). Scale bar of 10 pm.

[0044] Figure 10: Embodiment of DOX release along time from 1 mM of LUVs incorporated in a hydrogel of 5% not (A) and RGDS-functionalized (B) hyaluronic acid.

[0045] Figure 11: Embodiment of percentage of GL42 cells viability in the presence of different concentrations of DOX (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 and 100 pM) after 1, 2, 3 and 7 days of culture.

[0046] Figure 12: Embodiment of metabolic activity of GL42 cells when incubated with different concentrations of LUVs (10, 7.5, 5, 2.5 and 1 mM). Control (Ctr) 1 is obtained from the cells culture only with medium (without treatment) and Ctr 2 is when cells are exposed to a mixture of medium and PBS added in the 10 mM LUVs condition. The symbol (*) denote significant differences versus the Ctr 2: **** p< 0.0001.

[0047] Figure 13: Embodiment of the metabolic activity of GL42 cells incubated with different concentrations of LUVs (0.05, 0.15, 0.25, 0.5 and 1 mM) incorporating DOX. Control (Ctr) 1 is obtained from the cells culture only with medium (without treatment) and Ctr 2 is when cells are exposed to a mixture of medium and PBS added in the 1 mM LUVs condition. The symbol (*) denote significant differences versus the Ctr 1 and 2: ****p< 0.0001; **p=0.0007; *p=0.0006.

[0048] Figure 14: Embodiment of the metabolic activity of GL42 cells seeded on hyaluronic acid (A) hydrogels incorporating or not 150 pM of empty LUVs (HA+LUVs) or encapsulating 4 pM of DOX (HA+LUVs/DOX). The symbol (*) denote significant differences versus the different conditions: ****p<0.001; ***p=0.07; **p=0.06; *p<0.05. [0049] Figure 15: Embodiment of live/dead results of GL42 cells cultured on 48-well plates (Ctr) or seeded on HA, HA with 150 mM of DPPC, HA with DPPC/DOX with 4 mM of encapsulated DOX, HA functionalized, HA functionalized with 150 mM of DPPC and HA functionalized with 0.1 mM of DOX in DPPC liposomes hydrogels. Scale bar: 200 pm.

[0050] Figure 16: Embodiment of the metabolic activity of GL42 cells seeded on RGDS- functionalized hyaluronic acid (RGDS-HA) hydrogels incorporating or not 150 mM of empty LUVs (RGDS-HA+LUVs) or encapsulating 0.1 pM of DOX (RGDS-HA+LUVs). The symbol (*) denote significant differences versus the control: ****p<0.001.

[0051] Figure 17: Embodiment of the metabolic activity of GL42 cells (5000 cells/well) in the presence of 540 ng/mL of RGDS after 1, 3, 7 and 10 days of culture.

[0052] Figure 18: Embodiment of the metabolic activity of GL42 cells (5000, 8000 and 25000 cells/well of 48-well plates) in the presence of RGDS after 2 (A) and 7 (B) days of culture. Control 1 (Ctr 1) is of cells cultured only with medium and Ctrl 2 is of cells treated with medium without FBS for 24 h, being afterwards, replaced with fresh medium. The symbol (*) denote significant differences versus the Ctr 1: ****p< 0.0001; *p<0.05.

[0053] Figure 19: Illustration of fluorescence microscope images of live/dead staining with Calcein AM/PI (green: live cell; red: dead cell) of GL42 cells cultured with RGDS for 2 and 7 days. Ctr 1 refers to GL42 cells without treatment and Ctr 2 is GL42 cells treated for 24 h with medium without FBS. Scale bar: 200 pm.

[0054] Figure 20: Embodiment of the metabolic activity (A), proliferation (B) and MMP2 production (C) by GL42 cells (5xl0⁴ cells/well of 48-well plates). The symbol (*) denote significant differences versus the different days: ****p< 0.0001.

[0055] Figure 21: Embodiment of RGDS release (%) from the functionalized hydrogel in the presence of MMP-2 for 24, 48 and 72 h.

[0056] Figure 22: Embodiment of GL42 cells (A) and human immortalized astrocytes hTERT/E6/E7 cell line (B) metabolic activity when cultured on 48-well culture plates or seeded on RGDS-functionalized hyaluronic acid (RGDS-HA) without or with 150 mM LUVs (RGDS-HA+LUVs) not or encapsulating 0.1 mM DOX (RGDS-HA+LUVs/DOX). The symbol (*) denote significant differences versus the control: ****p<0.001; *p<0.05. [0057] Figure 23: Illustration of fluorescence microscope images of live/dead staining with Calcein AM/PI (green: live cell; red: dead cell) of GL42 cells seeded on the bottom of the well (Ctr) and on RGDS-functionalized hyaluronic acid (RGDS-HA) with 150 mM of empty LUVs (RGDS-HA+LUVs) or incorporating DOX (0.1 mM) and in co-culture with hTERT cells seeded on cell culture inserts. Scale bar: 200 miti.

[0058] Figure 24: illustration of fluorescence microscope images of live/dead staining with Calcein AM/PI (green: live cell; red: dead cell) of human immortalized astrocytes hTERT/E6/E7 cell line seeded on cell culture inserts (Ctr) and in co-culture with GL42 cells cultured on RGDS-functionalized hyaluronic acid (RGDS-HA) hydrogels containing or not 150 mM of liposomes (RGDS-HA+LUVs) or with liposomes incorporating 0.1 mM DOX (RGDS-HA+LUVs). Scale bar: 200 pm.

DETAILED DESCRIPTION

[0059] The present disclosure relates to a biocompatible hydrogel composition for use in the treatment or therapy of a solid tumor comprising a hyaluronic acid polymer; one or more peptide sequence, wherein the peptide sequence is an inhibitor of an extracellular matrix protein, namely an inhibitor of fibronectin, tenascin C, vitronectin, laminin or collagen; and liposomes, wherein the liposomes are arranged to physically crosslink the hyaluronic acid polymer; or hyaluronic acid and at least a sequence 90% identical to the sequences of the following list: SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, or mixtures thereof, wherein the peptide sequence is chemically bound to the hyaluronic acid polymer.

[0060] The present disclosure also relates to a hydrogel composition for use in the treatment of glioblastoma comprising hyaluronic acid, at least one peptide sequence, and liposomes, wherein the liposomes encapsulate an anti-cancer drug.

[0061] In an embodiment, large unilamellar liposomes (LUVs) of a phospholipid were produced and characterized, preferably LUVs of l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC). DPPC is a saturated phospholipid [PC (16:0/16:0)] and, consequently, its tails are straight and can pack tightly, producing liposomes with a decreased fluidity. Therefore, when the liposomes interact with cancer cell membranes (exchange of lipids, adsorption or binding, internalization or fusion of liposomes with the cell membrane), there is a decrease of their fluidity. The membrane of GB cells present a high content in unsaturated fatty acids and, consequently, fluidity, which may enhance tumor cell motility and invasion [44]

[0062] In another embodiment, LUVs incorporating or not DOX were prepared by the thin film hydration method followed by extrusion [45, 46] The non-encapsulated DOX was removed by column chromatography, but other means can be used and are known in the state of the art, such as centrifugation (already used). Table 2 shows that DPPC LUVs incorporating DOX presented a slightly higher size, polydispersity index (PDI) and zeta potential than empty liposomes. In an embodiment, the homogeneous suspension of LUVs was stable for the period of time considered (10 days), presenting at the end of the experiment a size of 124.4 ± 5.9 nm, a PDI of 0.111 ± 0.035 and a zeta potential of -3.34 ± 1.63 (Figure 1).

Table 2: Size, PDI and zeta potential of liposomes incorporating or not DOX

Sample Size (nm) PDI Zeta Potential (mV)

LUVs 114.7 ± 2.3 0.076 ± 0.023 -1.38 ± 0.99

LUVs with DOX 121.7 ± 4.7 0.164 ± 0.041 -2.43 ± 1.33

[0063] In an embodiment, scanning transmission electron microscopy (STEM) images (Figure 2) demonstrated that liposomes incorporating or not DOX have a round shape and size values that are in agreement with the dynamic light scattering (DLS) results (Table 2). Moreover, HPLC analysis demonstrated that LUVs were able to encapsulate 68.2 ± 13.5 pg of DOX for a phospholipid concentration of 1 mM.

[0064] In an embodiment, the thermal stability of LUVs was assessed using differential scanning calorimetry (DSC) analysis. The thermograms of liposomes incorporating or not DOX are presented in Figure 3. DPPC liposomes presented an endothermic peak at 55.80 ± 0.46 °C with an onset temperature of 46.53 ± 3.05 °C. The incorporation of DOX into liposomes lead to the presence of a more defined endothermic peak at 51.46 ± 0.35 °C with an onset temperature of 49.11 ± 0.14 °C.

[0065] Methods for the alignment of sequences for comparison are well known in the art. Such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch [47] to find the global (over the whole the sequence) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm [48] calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package [49] Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. The sequence identity values, which are indicated in the present subject matter as a percentage were determined over the entire amino acid sequence, using BLAST with the default parameters.

[0066] In an embodiment, the hyaluronic acid was functionalized with the short peptide sequence RGDS using the carbodiimide chemistry. The peptide was synthesized by solid-phase peptide synthesis using Fmoc chemistry in an automated peptide synthesizer (CS Bio), followed by its characterization using a HPLC with MS detector. When needed, the peptide was purified by preparative HPLC and the salt of trifluoroacetic acid (TFA) was exchanged by acid removal filter (VariPure™ IPE Ion Pair Extraction). To link the peptide to hyaluronic acid, first the activation of the carboxyl groups of the hyaluronic acid by N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC)/N-hydroxysuccinimide (NHS) chemistry, at pH 6.5, was performed and, then, the peptide was added. The reaction of the amine group of the peptide with the activated HA led to the formation of amide bonds. After overnight incubation at 4 °C, the resulting solution was dialyzed to remove unreacted compounds. Afterwards, the functionalized hyaluronic acid was freeze-dried. Nuclear Magnetic Resonance (NMR) spectroscopy and Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy analyses were performed to confirm the chemical modifications. Figure 4 presents the ¹H-NMR spectra of RGDS-functionalized-hyaluronic acid as well as of the natural polymer and peptide. The acetamido moiety of the N-acetyl-D-glucosamine residue of the hyaluronic acid was located at 6=2.18 ppm when it is used without further processing. Its linking to RGDS presented fingerprints of the natural polymer skeletal signals at 6=3.5-4.0 ppm. There was also a small shift of CH3 to 2.03 ppm. All peaks of the RGDS were observed in the RGDS-functionalized hyaluronic acid spectrum. The main evidence was the alteration of the shift of the a-C of arginine from 4.24 ppm in RGDS to 3.5 ppm when linked to the polymer. Two of the three CH2 of arginine also have a big shift in the RDGS-functionalized hyaluronic acid. In addition, the ¹³C-NMR spectrum confirms the RGDS binding since the quaternary C of arginine appears at 160 ppm.

[0067] Figure 5 represents an embodiment of the results of ATR-FTIR analysis of the hyaluronic acid functionalized with RGDS. The spectrum of the hyaluronic acid functionalized with the peptide presents the mains groups characteristic of the polymer as well as the amides I and II vibration frequency bands at 1630 and 1535 cm^-1, respectively, of RGDS, proving the successful chemical conjugation of hyaluronic acid with RGDS.

[0068] In an embodiment, the rheological properties of the hyaluronic acid hydrogels were also assessed, at 25 °C and 37 °C. The storage (elastic, G') and loss (viscous, G") moduli of 1%, 2.5% and 5% (w/v) of hyaluronic acid hydrogels are presented in Figure 6 and Table 3. The G' and G” moduli increased for higher concentrations of hyaluronic acid. However, for each concentration, the G' and G” values decreased with the increase of the temperature. Since the mechanical properties of hyaluronic acid at 5% (w/v) presents more similarities with the rheological properties of the brain tissue [2], this concentration was selected to perform the remaining assays.

[0069] In the state of the art, the rheological properties may be measured by many methods. In an embodiment, the rheological properties were measured using a Kinexus Prot rheometer (Malvern) coupled with a 20 mm geometry plate. Frequency sweeps were obtained using a predefined shear strain of 1%. Amplitude sweep strain- controlled tests at a frequency of 1 Hz were performed to determine the LVR. Subsequent frequency sweep tests were performed between 0.1-100 Hz using a constant strain within the LVR of 1 Hz to determinate G', phase shift (d) and G”.

Table 3. Values of elastic (G') and viscous (G") moduli of hydrogels containing 1%, 2.5% and 5% (w/v) hyaluronic acid (HA) at 25 °C and 37 °C.

HA (%w/v) T (°C) G'(kPa) G” (kPa)

25 0.068 ± 0.016 0.035 ± 0.015

1

37 0.065 ± 0.015 0.047 ± 0.010

25 0.478 ± 0.011 0.267 ± 0.081

2.5

37 0.347 ± 0.015 0.204 ± 0.079

25 3.831 ± 0.905 1.402 ± 0.230

5

37 2.529 ± 0.653 1.059 ± 0.169

[0070] Figure 7 represents an embodiment of the rheological properties of the hyaluronic acid functionalized or not with RGDS and incorporating or not 150 mM of LUVs. The values of G' and G” are presented in Table 4. The functionalization of hyaluronic acid decreased the values of G' and G”. Moreover, the addition of 150 pM LUVs in the hydrogel and the increase of temperature slightly decreased the mechanical properties of the hydrogel.

Table 4. Values of elastic (G') and viscous (G") moduli of hydrogels of hyaluronic acid(HA); hydrogels of HA functionalized with RGDS (HA-RGDS); hydrogels of HA incorporating LUVs (HA+LUVs); and hydrogels of HA functionalized with RGDS and incorporating LUVs (HA-RGDS+LUVs) at 25 °C and 37 °C.

Formulation T (°C) G'(kPa) G"(kPa)

25 3.831 ± 0.905 1.402 ± 0.230

HA

37 2.529 ± 0.653 1.059 ± 0.170

25 3.605 ± 1.569 1.411 ± 0.560

HA+LUVs

37 2.889 ± 1.095 1.205 ± 0.419

25 0.938 ± 0.248 0.525 ± 0.097

HA-RGDS

37 0.825 ± 0.116 0.427 ± 0.122

25 0.818 ± 0.340 0.476 ± 0.160

HA-RGDS+ LUVs

37 0.777 ± 0.198 0.461 ± 0.139

[0071] In an embodiment, the thermal properties of the different formulations were assessed using DSC (Figure 8). The thermograms of hyaluronic acid functionalized or not with RGDS show one endothermic peak at 23.37 ± 0.20 °C and 26.56 ± 2.75 °C and one exothermic peak at 210.18 ± 3.64 °C and 233.76 ± 0.43 °C, respectively. Hyaluronic acid with empty liposomes and incorporating DOX presents an endothermic peak at 26.91 ± 4.88 °C and 25.07 ± 1.62 °C and exothermic peaks at 230.98 ± 1.37 °C and 233.17 ± 1.45 °C in its thermogram (Figure 8). RGDS-functionalized hyaluronic acid incorporating empty LUVs or incorporating DOX thermograms show an endothermic peak at 28.49 ± 2.38 °C and 21.35 ± 2.14 °C and exothermic peaks at 219.10 ± 1.82 °C and 233.17 ± 0.16 °C (Figure 8). Consequently, LUVs containing or not the anticancer drug did not alter the thermal properties of the polymer subjected or not to a chemical procedure.

[0072] Figure 9 shows and embodiment of the morphology of the different hydrogels. The hyaluronic acid hydrogel presented a more porous morphology than hydrogels of RGDS-functionalized hyaluronic acid, which presents a more compact morphology. Since formulations were prepared in phosphate buffered saline (PBS) buffer (pH 7.4), salts at their surface were observed in the freeze-dried samples.

[0073] Figure 10 shows an embodiment of the release of DOX from LUVs incorporated in hydrogels of hyaluronic acid functionalized or not with RGDS. In an embodiment, the RGDS-functionalized hyaluronic acid hydrogels incorporating liposomes released a higher amount of DOX than hydrogels with a similar composition, but using the natural polymer without chemical modification.

[0074] In an embodiment, to determine the biological performance of the hydrogels, two cell lines were used, namely the human primary GB GL42 cell line and the human immortalized astrocytes hTERT/E6/E7 cell line.

[0075] First, the half maximal inhibitory concentration (IC50) of DOX for GL42 cells was determined. Figure 11 presents the dose-response curves obtained in this experiment and Table 5 presents the IC50 values for the different time points.

Table 5. IC50 of DOX for GL42 cells at different time points (1, 2, 3 and 7 days).

Day IC₅₀ (mM)

1 3.909 + 1.160

2 0.162 + 0.037

3 0.128 + 0.021

7 0.134 + 0.025

[0076] In an embodiment, the metabolic activity of cells in the presence of the liposomes was also evaluated (Figure 12). From comparison of the two controls (controls 1 and 2 are related with the culture of cells only in RPMI 1640 medium and in the medium diluted with the maximum amount of PBS present in the condition with the highest concentration of LUVs, respectively), it is possible to verify that the addition of LUVs in PBS does not affect the cells metabolic activity. Figure 12 also demonstrates that concentrations of LUVs smaller than 5 mM are not cytotoxic for the cells. [0077] In an embodiment, the GL42 metabolic activity was assessed in the presence of different concentrations of liposomes incorporating DOX. Figure 13 shows an embodiment of the results. Control 1 and Control 2 are cells seeded in culture media and cells cultured in media supplemented with the maximum amount of PBS in the condition of 1 mM LUVs, respectively. In the presence of the liposomes incorporating DOX, the cells metabolic activity significantly decreases after 24 h of culture, being practically null for the remaining days. This demonstrates the effectiveness of the liposomes formulation as an anticancer therapy.

[0078] In an embodiment, GL42 cells seeded in hyaluronic acid hydrogels containing or not 150 mM of LUVs (this concentration was selected considering the maximum amount of liposomes that can be used according to DOX IC50) present an increase of their metabolic activity proving that these formulations are non-cytotoxic (Figure 14). Conversely, the incorporation of LUVs with 4 mM of DOX in the hyaluronic acid hydrogels led to a significantly decrease of the cells metabolic activity after the first 24 h of culture (Figure 14). Live/dead assays (Figure 15) confirmed the results of GL42 cells metabolic activity, presenting an increase in the number of live cells for hyaluronic acid hydrogels containing or not liposomes. However, the inclusion of DOX in the formulations led to a significant increase of the dead cells. Indeed, in the last time points no viable cells were present (Figure 15).

[0079] In an embodiment, despite the non-cytotoxicity of hyaluronic-based hydrogels, the GL42 cells seeded on RGDS-functionalized hyaluronic acid hydrogels incorporating or not liposomes, encapsulating or not DOX, presented a not expected decrease in their metabolic activity (Figure 16) and viability (Figure 15). Consequently, these formulations present cytotoxicity, and therefore, the cytocompatiblity of the free peptide was evaluated. The GL42 metabolic activity in the presence of 540 ng/mL of RGDS (maximum peptide concentration present in the hydrogel formulations used in the in vitro assays) is presented in Figure 17. RGDS was not cytotoxic. However, as the presence of fetal bovine serum (FBS) in the medium can influence the results obtained, due to the presence of a high amount of proteins, other experiment was performed. Two controls were performed, namely control 1 was of cells cultured only with medium (with FBS and without peptide) and control 2 was of cells treated with medium without FBS for 24 h, being afterwards, replaced with fresh medium. As can be observed in Figure 18, cells cultured in the presence of RGDS showed a metabolic activity smaller than control 1 but similar to the control 2. Consequently, the decrease of their metabolic activity may be due to the absence of FBS in the medium instead of RGDS. However, after 7 days, cells were able to proliferate reaching the control 1 values. These results are also confirmed in the live/dead images (Figure 19). Consequently, RGDS in solution does not affect GL42 cells metabolic activity and viability.

[0080] Despite RGDS is not cytotoxic if added dissolved in the medium to the GL42 cells adhered to the bottom of the wells, we hypothesized that a different scenario can be obtained if the peptide is released from the hydrogel where the cells are adhered. Indeed, the peptide binding to the hydrogel can be broken by enzymes released from GL42 cells. GB cells present the ability of secretion of several proteolytic enzymes for the destruction of the ECM, such as matrix metalloproteinases (MMPs) [50] For instance, MMP-2 and MMP-9 are involved in the progression of GB, but in normal brain their expression is not observed [50] Consequently, the expression of MMP-2 by GL42 cells was investigated.

[0081] The GL42 cells metabolic activity, proliferation, and expression of MPP-2 is present in Figure 20. In Figure 20A, it is possible to observe that cells metabolic activity increased until 7 days and remained similar after 10 days of culture. Conversely, they were able to proliferate during all the experimental period of time considered (Figure 20B). Finally, the MPP-2 expression by GL42 cells (Figure 20C) also increased until the end of the experiment, reaching a final value of 18.45 ± 0.46 ng/mL (p<0.0001).

[0082] The MMP-2 ability to cleave the RGDS-hyaluronic acid chemical binding was also evaluated. Figure 21 shows the RGDS release from the hydrogel in the presence of MMP-2. Consequently, the cytotoxicity of the RGDS-functionalized hydrogel can be related with the released peptide which GB cells can bind to. In an embodiment, the inhibition of binding of GB cells to ECM can induce an anchorage-dependent apoptosis, anoikis. In another embodiment, the RGDS peptide prevents focal adhesion assembly and disrupts the integrity of the cytoskeleton induced by fibronectin engagement with integrin. Moreover, RGD motifs can be internalized by cells and activate caspases and survivin, leading to apoptosis.

[0083] To evaluate the toxicity of this strategy for healthy cells, a co-culture of GB cells and astrocytes cells is performed. The GL42 cells were seeded on RGDS-hyaluronic acid hydrogels incorporating or not empty LUVs or encapsulating DOX and the astrocytes are seeded in cell culture inserts. As demonstrated in Figure 22A, there is a decrease in the GL42 cells metabolic activity in the presence of the different formulations. Live/dead results also confirm the decrease of cells viability (Figure 23). The immortalized human astrocytes hTERT/E6/E7 cell line metabolic activity in the presence of the several formulations was similar to the control at the different time points (Figure 22B). These data were confirmed by live/dead assays (Figure 24). Indeed, the confocal microscopy images of the live/dead staining showed that astrocytes remained viable for all time points (Figure 24). Consequently, the developed strategy is not cytotoxic for astrocytes.

[0084] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0085] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above described embodiments are combinable.

[0086] The following claims further set out particular embodiments of the disclosure.

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Claims

C L A I M S

1. A hydrogel composition for use in the treatment or therapy of a solid tumor comprising: a hyaluronic acid polymer; one or more peptide sequence, wherein the peptide sequence is an inhibitor of an extracellular matrix protein; and liposomes, wherein the liposomes are arranged to physically crosslink the hyaluronic acid polymer; or hyaluronic acid and at least a peptide sequence 90% identical to the sequences of the following list: SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, or mixtures thereof, wherein the peptide sequence is chemically bound to the hyaluronic acid polymer.

2. The hydrogel composition for use according to the previous claim wherein the liposomes encapsulate an anti-cancer drug.

3. The hydrogel composition for use according to any of the previous claims wherein the extracellular matrix protein is selected from a list comprising: fibronectin, tenascin C, vitronectin, laminin, collagen or mixtures thereof.

4. The hydrogel composition for use according to any of the previous claims wherein the one or more peptide sequence comprises at least a sequence 90% identical to the sequences of the following list: SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, or mixtures thereof.

5. The hydrogel composition for use according to any of the previous claims wherein the one or more peptide sequence comprises at least a sequence 95% identical to the sequences of the following list: SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, or mixtures thereof.

6. The hydrogel composition for use according to any of the previous claims wherein the one or more peptide sequence comprises at least a sequence identical to the sequences of the following list SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, or mixtures thereof.

7. The hydrogel composition according to any of the previous claims for use in the treatment or therapy of glioblastoma.

8. The hydrogel composition for use according to any of the previous claims comprising:

1-20% (wt/Vhy_drogei) of hyaluronic acid, preferably 2-10% (wt/Vh_ydro_gei) of hyaluronic acid;

100 to 10000 ng/mL of the one or more peptide sequence, preferably 400 to 1000 ng/mL of the one or more peptide sequence; and

5-10000 mM of liposomes, preferably 50-1000 pM of liposomes; wherein the liposomes encapsulate 0.1-10000 pM of an anti-cancer drug, preferably 1-1000 pM of anti-cancer drug.

9. The hydrogel composition for use according to any of the previous claims wherein the one or more peptide sequence is chemically bound to the hyaluronic acid polymer.

10. The hydrogel composition for use according to any of the previous claims wherein the bound between the hyaluronic acid and the peptide sequence is cleavable by proteolytic enzymes, preferably matrix metalloproteinases.

11. The hydrogel composition for use according to any of the previous claims wherein the liposomes are physically linked to hyaluronic acid by electrostatic interactions.

12. The hydrogel composition for use according to any of the previous claim wherein the liposomes are small unilamellar liposomes, large unilamellar liposomes, or mixtures thereof.

13. The hydrogel composition for use according to any of the previous claims wherein the liposomes have a size ranging from 25 to 400 nm, preferably from 110 to 130 nm.

14. The hydrogel composition for use according to any of the previous claims wherein the liposomes are large unilamellar liposomes of l,2-dipalmitoyl-sn-glycero-3- phosphocholine, phosphatidylcholine, phosphatidylserine, glycerophosphocholine, glycolipids, hydrogenated PC, cholesterol, PEGylated phospholipids, PEGylated phospholipids containing a functional group at the polymeric end, 1,2-distearoyl- sn-glycero-3-phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine, and/or cholesteryl hemisuccinate.

15. The hydrogel composition for use according to any of the previous claims 2-14 comprising 0.1-10000 mM of the anti-cancer drug.

16. The hydrogel composition for use according to any of the previous claims 2-15 wherein the anti-cancer drug is selected from a list comprising drugs used in therapeutic, immune checkpoint inhibitors, natural compounds, repurposing or promising drugs, or mixtures thereof.

17. The hydrogel composition for use according to any of the previous claims 2-16 wherein the anti-cancer drug is selected from a list comprising T-lymphocyte- associated antigen 4, programmed death 1, programmed death-ligand 1, indolamine-2, 3-dioxygenase, arginase 1, dihydroartemisinin, paclitaxel, lomustine, carmustine, bevacizumab, doxorubicin, CXCR3-A inhibitors, mefloquine, ritonavir, ribavirin, chlorpromazine, temozolomide, ivermectin, or mixtures thereof.

18. The hydrogel composition for use according to any of the previous claims 2-17 wherein the anti-cancer drug is doxorubicin.

19. The hydrogel composition for use according to any of the previous claims wherein the hydrogel is injectable.

20. The hydrogel composition for use according to any of the previous claims wherein the hydrogel is administrable with a neurosurgical instrument, preferably a spatula.

21. The hydrogel composition for use according to any of the previous claims wherein the hydrogel is administrable by an in-situ injection or intra-tumor injection.

22. The hydrogel composition for use according to any of the previous claims wherein the storage modulus at 37 °C ranges from 0.5 to 3.5 kPa.

23. The hydrogel composition for use according to any of the previous claims wherein the storage modulus at 25 °C ranges from 0.4 to 5 kPa.

24. A pharmaceutical composition for use in the treatment or therapy of a solid tumor, preferably glioblastoma, comprising a therapeutically effective amount of a hydrogel described in any of the previous claims and a pharmaceutically acceptable excipient/carrier.